Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure

Details

• Alternative Title:
  Chem Res Toxicol
• Personal Author:
  Dafferner, Alicia J. ; Schopfer, Lawrence M. ; Xiao, Gaoping ; Cashman, John R. ; Yerramalla, Udaya ; Johnson, Rudolph C. ; Blake, Thomas A. ; Lockridge, Oksana
• Description:
  Nerve agents and organophosphorus pesticides make a covalent bond with the active site serine of acetylcholinesterase (AChE), resulting in inhibition of AChE activity and toxic symptoms. AChE in red blood cells (RBCs) serves as a surrogate for AChE in the nervous system. Mass spectrometry analysis of adducts on RBC AChE could provide evidence of exposure. Our goal was to develop a method of immunopurifying human RBC AChE in quantities adequate for detecting exposure by mass spectrometry. For this purpose, we immobilized 3 commercially available anti-human acetylcholinesterase monoclonal antibodies (AE-1, AE-2, and HR2) plus 3 new monoclonal antibodies. The monoclonal antibodies were characterized for binding affinity, epitope mapping by pairing analysis, and nucleotide and amino acid sequences. AChE was solubilized from frozen RBCs with 1% (v/v) Triton X-100. A 16 mL sample containing 5.8 μg of RBC AChE was treated with a quantity of soman model compound that inhibited 50% of the AChE activity. Native and soman-inhibited RBC AChE samples were immunopurified on antibody-Sepharose beads. The immunopurified RBC AChE was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. The aged soman-modified PheGlyGluSerAlaGlyAlaAlaSer (FGESAGAAS) peptide was detected using a targeted analysis method. It was concluded that all 6 monoclonal antibodies could be used to immunopurify RBC AChE and that exposure to nerve agents could be detected as adducts on the active site serine of RBC AChE.
• Subjects:
  Acetylcholinesterase Article Erythrocytes Humans Immunoprecipitation Mass Spectrometry Nerve Agents
• Source:
  Chem Res Toxicol. 2017; 30(10):1897-1910.
• Pubmed ID:
  28892361
• Pubmed Central ID:
  PMC5646370
• Document Type:
  Journal Article
• Funding:
  P30 CA036727/CA/NCI NIH HHS/United States
• Volume:
  30
• Issue:
  10
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:4d592b4a7165467392c451ff9dd835907e299aec3a464a28de5433ef767a7e18
• Download URL:
  https://stacks.cdc.gov/view/cdc/59805/cdc_59805_DS1.pdf
• File Type:
  [PDF - 4.79 MB ]